Camila Gewehra, Mariane Arnoldi da Silvab, Gabriela Trevisan dos Santosb, Mateus
Fortes Rossatob, Carine Cristiane Drewesc, Andréia Martini Pazinib, Gustavo Petri
Guerrab, Maribel A. Rubina,b,c, Juliano Ferreiraa,b,c,*
a
Programa de Pós-Graduação em Farmacologia, bPrograma de Pós-Graduação em
Ciências Biológicas: Bioquímica Toxicológica, cDepartment of Chemistry, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.
*Author for Correspondence: Department of Chemistry, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil; FAX (5555) 3220-8978, Tel (5555) 32208053. E-mail: [email protected]
Abstract: Polyamines (putrescine, spermidine and spermine) are important
endogenous regulators of ion channels, such as vanilloid (TRPV1), glutamatergic (NMDA or AMPA/kainate) and acid-sensitive (ASIC) receptors. In the present study, we have investigated the possible nociceptive effect induced by polyamines and the mechanisms involved in this nociception. The subcutaneous (s.c.) injection of capsaicin, spermine, spermidine or putrescine produced nociception with ED50 of
0.16 (0.07-0.39) nmol/paw, 0.4 (0.2-0.7) µmol/paw, 0.3 (0.1-0.9) µmol/paw and 3.2 (0.9-11.5) µmol/paw, respectively. The antagonists of NMDA (MK801, 1 nmol/paw), AMPA/kainate (DNQX, 1 nmol/paw) or ASIC receptors (amiloride, 100 nmol/paw) failed to reduce the spermine-trigged nociception. However, the TRPV1 antagonists capsazepine or SB366791 (1 nmol/paw) reduced spermine-induced nociception, with inhibition of 81±10 and 68±9%, respectively. The previous desensitization with resiniferatoxin (RTX) largely reduced the spermine-induced nociception and TRPV1 expression in the sciatic nerve, with reductions of 82±9% and 67±11%, respectively. Furthermore, the combination of spermine (100 nmol/paw) and RTX (0.005 fmol/paw), in doses which alone were not capable of inducing nociception, produced nociceptive behaviors. Moreover, different concentrations of spermine (3-300 µM) enhanced the specific binding of [3H]-RTX to TRPV1 receptor. Altogether,
polyamines produce spontaneous nociceptive effect through the stimulation of TRPV1, but not of ionotropic glutamate or ASIC receptors.
Perspective: This article shows that polyamines could be important peripheral
modulators of pain, especially during inflammatory processes when local polyamine levels are increased.
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Introduction
Polyamines putrescine (1,4-butane diamine), spermidine (N-(3-aminopropyl)- 1,4-butane diamine) and spermine (N,N0-bis(3-aminopropyl)-1,4-butane diamine) are aliphatic amines initially formed by decarboxylation of ornithine, a reaction catalysed by the enzyme ornithine decarboxylase.31 Known functions of polyamines include
interactions with several biomolecules, specially ion channels. 8,26,34,35
In fact, various functions of the central nervous system (CNS) are related with polyamine actions on ion channels.30 Several in vitro and in vivo evidences have
demonstrated that polyamines can modulate N-methyl-D-aspartate (NMDA), α- amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or kainite receptors in CNS.30,35 It is also already known that polyvalent cations, such as spermine, are
modulators of ASIC receptor activity.3 Interestingly, an in vitro study has
demonstrated that intracellular and extracellular polyamines can modulate the vanilloid receptor TRPV1 activity, a member of the transient receptor potencial (TRP) channel family.2 These findings suggest that polyamine could also regulate the
function of ion channels in other tissues.
TRPV1, ASIC and glutamatergic NMDA or kainate receptors are expressed by a subset of peripheral pain-sensing neurons.5,6,24 When such receptors are exposed
to tissue damaging stimuli, these channels become permeable to Na+ and Ca2+ ions,
causing, in turn, neuronal depolarization and recognition as pain stimulus. Sensory neuron firing transmits these pain signals towards the central nervous system, evoking at the same time a variety of local tissue responses.7 The peripheral
administration of vanilloid, ASIC or glutamate receptor agonists results in nociceptive behavior in mice and rats.4,12,21,28,37 On the other hand, peripheral administration of
inflammatory pain.10,21,29 Of note, the levels of polyamines are increased in tissue and
synovial fluid of patients with arthritis,36 suggesting polyamines could mediate
inflammatory pain.
Therefore, the present study aimed to assess the role of peripheral polyamines in nociception in vivo and investigate the role of glutamatergic NMDA and AMPA/kainite as well as ASIC and TRPV1 receptors in this action.
Methods Animals
Male Swiss mice (30–35 g) maintained at 22±2ºC with free access to water and food, under a 12:12 h light: dark cycle, were used. Animals were acclimatized in the laboratory for at least 2 h before testing and were used once throughout the experiments. All experiments were conducted in accordance to the ethical guidelines of the International Association for the Study of Pain,39 the National Institutes of
Health guide for the care and use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The number of animals and the nociceptive stimulus were the minimum necessary to demonstrate the consistent effects of drug treatments. This study was approved by the Committee on the Use and Care of Laboratory Animals of our university (no. 23081.012331/2009-81).
Algogen-induced nociceptive responses
The procedure used was similar to that previously described by Ferreira et al and Oliveira et al.12,22 A volume of 20 µl of capsaicin (0.01–3 nmol/paw), glutamate (10000 nmol/paw), putrescine (1000-10000 nmol/paw), spermidine (100-2000 nmol/paw) or spermine (100–2000 nmol/paw) was injected subcutaneously (s.c.)
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under the surface of the right hind paw. Separate groups of animals received s.c. injection of the appropriated vehicle phosphate-buffered saline (PBS) (137 mM NaCl, 27 mM KCl and 10 mM phosphate buffer) or PBS plus ethanol 0.25% for capsaicin. Animals were placed individually in chambers (transparent glass cylinders of 20 cm in diameter) and were adapted for 20 min before treatment. After challenge, mice were observed individually for 5 min and the amount of time spent licking the injected paw was timed with a chronometer and was considered as indicative of nociception. The dose and the time of administration of drugs were based on pilot studies.
Involvement of NMDA, AMPA/kainate, TRPV1, ASIC receptors in spermine- induced nociception
In order to assess the involvement of NMDA, AMPA/kainate or TRPV1 receptors in the nociceptive responses induced by spermine (1000 nmol/paw), animals were co-administered with the selective NMDA receptor antagonist MK 801 (1 nmol/paw), AMPA/kainate receptor antagonist DNQX (1 nmol/paw), acid-sensitive ion channel (ASIC) blocker amiloride (100 nmol/paw), TRPV1 receptor antagonist capsazepine (0.1–1 nmol/paw), the seletive TRPV1 receptor antagonist SB366791 (1 nmol/paw) or the TRPV1 agonist resiniferatoxin (RTX; 0.005 pmol/paw). The choice of the dose of antagonists was based on previous data described in literature.
4,12,13,21,22
Control animals received a similar volume of vehicle (20 µl/paw). To confirm the efficacy of the antagonist tested, we tested the effect of MK801 (1 nmol/paw) and DNQX (1 nmol/paw) co-administration with glutamate (10000 nmol/paw), as well as capsazepine (1 nmol/paw) and SB366791 (1 nmol/paw) co- administration with capsaicin (1 nmol/paw).
Desensitization of TRPV1 positive fibers in spermine-induced nociception
To further explore the role of TRPV1 positive (TRPV1+) fibers in the nociceptive induced by spermine, the animals were submitted to a systemic desensitization protocol with RTX as previously described by Hsieh et al.15 Animals
were anesthetized with isoflurane and received systemic administration of RTX (50 µg/Kg) or the vehicle alone (0.5% ethanol, 0.5% Tween-80, PBS). After 7 days, animals were submitted to a subcutaneous injection of spermine (1000 nmol/paw, s.c.), capsaicin (1 nmol/paw, used as a positive control) or vehicle (0.5 % ethanol, 0.5% Tween-80 and PBS) (20 µl/paw).
TRPV1 receptor expression following systemic RTX treatment
The effect of the systemic treatment with RTX on the expression of TRPV1 receptors was assessed by Western blot analysis. The assay was carried out as previously described by Andre et al with minor modifications.1 The sciatic nerves
were quickly isolated and were homogenized in a lyses buffer containing 10 mM HEPES, pH 7.9, 10 M KCl, 2 M MgCl2, 0.1 M ethylenediamine tetraacetic acid
(EDTA), 1 M NaF, 10 µg/mL aprotinin, 10 M β-glicerolphosphate, 1 mM phenylmethanesulphonyl fluoride, 1 M DL-dithiothreitol (DTT) and 2 M of sodium orthovanadate. After centrifugation (3000 xg for 30 mim), the supernatant was collected. The protein content was determined by the method of Bradford (1976), using bovine serum albumin as a standard. Amounts of 30 µg of protein of sciatic nerve were mixed in load buffer (200 mM Tris, 10% glycerol, 2% SDS, 2.75 mM β- mercaptoethanol and 0.04% bromophenol blue) and boiled for 10 min. Proteins were separated in 10% sodium dodecyl sulphate–polyacrylamide gels (SDS–PAGE) and
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transferred to polyvinilidene difluoride membranes, according to the manufacturer’s instructions (Perkin Elmer, USA). The Ponceau staining served as a loading control. After staining the membranes were dried, scanned and quantified. Membranes were processed using a SNAP i.d. system (Millipore, USA), blocked with 1% BSA in TBS-T (0.05% Tween 20 in Tris-borate saline) and then incubated for 10 min, with specific antibodies to anti-TRPV1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:150 in TBS-T. Blots were washed three times with TBS-T followed by incubation with alkaline phosphatase-coupled secondary antibody (1:3000) for 10 min. The protein bands were visualized with 5-bromo-4-chloro-3-indolyl phosphate/p-nitro blue tetrazolium system (BCIP/NBT). Membranes were dried, scanned and quantified with Scion Image PC version of Macintosh compatible NIH image. The TRPV1 western blot had a faint background that was corrected in the image analysis.
[3H]-RTX Binding Assay
Binding assays were carried out as previously described by Andre et al, with minor modifications.1 To obtain membranes for the binding studies, spinal cords (a
rich source of vanilloid receptors) of mice were removed and disrupted with the aid of a tissue homogenizer in ice-cold buffer A, pH 7.4, which contained 5 mM KCl, 5.8 mM NaCl, 2 mM MgCl2, 0.75 mM CaCl2, 12 mM glucose, 137 mM sucrose, and 10 mM HEPES. The homogenate was firstly centrifuged for 10 min at 1000 xg in 4°C. The low-speed pellets were discarded, and the supernatants were further centrifuged for 30 min at 35,000 xg in 4°C. The resulting high-speed pellets, re-suspended in buffer A, were stored at -70°C until assayed. Binding assays were carried out in duplicate with a final volume of 500 µl, containing buffer A supplemented with 0.25
mg/ml bovine serum albumin, membrane (100 µg/protein) and 50 pM [3H]-RTX in
absence or presence of spermine (3 - 300 µM). For the measurement of non-specific binding, 100 nM non-radioactive RTX was included in some tubes. Assay mixtures were set up on ice, and the binding reaction was then initiated by transferring the assay tubes to a 37°C water bath. After a 60-minute incubation period, cooling the mixtures on ice terminated the binding reaction, and then 50 µL of bovine 1-acid glycoprotein (2 mg/mL) was added to each tube (to allow the detection of specific binding). Finally, the bound and free membranes of [3H]-RTX were separated by
centrifuging for 15 min at 20,000 xg in 4°C. The pellet was quantified using scintillation counting. Specific binding was calculated as the difference between the total and non-specific binding.
Drugs
The following drugs were used: spermine, spermidine, putrescine, MK801 (5S,10R-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), DNQX (6,7-dinitroquinoxaline-2,3-dione), glutamate, resiniferatoxin, capsazepine and capsaicin, SB366791 (N-(3-methoxyphenyl)-4-chlorocinnamide) (all from Sigma Chemical Company, St Louis, MO, U.S.A.). The stock solutions of the drugs were prepared in 1 mM phosphate-buffered solution (PBS) from Sigma (USA) pH 7.4 in siliconized plastic tubes, maintained at -20 ºC and diluted to the desired concentration just before use. Capsaicin, capsazepine, SB366791 and resinferatoxin stock solutions were prepared in absolute ethanol (90%) plus tween 80 (10%). For drug administration, the final concentration of ethanol and tween 80 did not exceed 0.5% and did not cause any detectable effect per se (results not shown). In addition, radio-labeled [3H]-resiniferatoxin was purchased from Perkin Elmer (USA).
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Statistical analysis
Results are presented as mean±s.e.m., except ED50 or ID50 values (i.e., the
dose of agonist necessary to produce 50% of the nocicpetive response relative to the maximum effect, or the dose of antagonists necessary to reduce the agonist response by 50% relative to the control value, respectively), which are reported as geometric means accompanied by their respective 95% confidence limits. ED50 and
ID50 values were calculated, using non-linear regression (sigmoidal dose-response
curve, variable slope) with the GraphPad Prism software. Emax (maximal effect) and
Imax (maximal inhibition) were calculated based on responses of the control group.
The statistical significance between the groups was assessed by means of unpaired Student’s t-test or one-way ANOVA followed by Dunnett’s or Student-Newmann- Keuls’ (SNK) test. P-values lower than 0.05 were considered as indicative of significance.
Results
Nociception elicited by peripheral polyamines in mice
The subcutaneously administration of capsaicin (0.01-3 nmol/paw), spermine spermidine or putrescine (100-10000 nmol/paw) in mice produced short-lasting and spontaneous nociception (Figure 1). This response started just after the administration and did not last more than 5 min. The calculated mean ED50 values
(and the 95% confidence limits) for capsaicin, spermine, spermidine or putrescine- induced licking were 0.16 (0.07-0.39) nmol/paw, 0.4 (0.2-0.7) µmol/paw, 0.3 (0.1-0.9) µmol/paw and 3.2 (0.9-11.5) µmol/paw, respectively. The maximal nociceptive response caused by capsaicin, spermine, spermidine and putrescine were 51±4,
70±10, 47±14 and 37±11 s, respectively. Since spermine and spermidine were more potent than putrescine in producing nociception, we have chosen a sub-maximal dose of spermine (1000 nmol/paw) to study some mechanisms involved in polyamine-induced nociception.
Lack of participation of NMDA, AMPA/Kainate and ASIC receptors in the spermine-induced nociception
The selective NMDA receptor antagonist MK-801 (1 nmol/paw) was not capable of altering spermine-induced nociception (1000 nmol/paw, Figure 2A). On the other hand, the co-administration of MK-801 (1 nmol/paw) partially, but significantly, reduced the nociception caused by glutamate (10000 nmol/paw, Figure 2B) with inhibition of 47±12% compared with the control group. The co-administration of the selective antagonist AMPA/kainate receptor DNQX was also not capable of altering spermine-induced nociception (1000 nmol/paw, Figure 2C), but partially reduced the nociception caused by glutamate (10000 nmol/paw, Figure 2D), with inhibition of 55±6% compared with the control group. Furthermore, we verified that the co-administration of amiloride (100 nmol/paw), an ASIC receptor blocker, was also not able to modify the nociception induced by spermine (1000 nmol/paw; Figure 3). These results suggest that ionotropic glutamate and ASIC receptors activation did not mediate spermine-induced nociceptive effect.
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We have further assessed the possible role of TRPV1 in spermine-induced nociception. The co-administration of the TRPV1 antagonist capsazepine (0.1–1 nmol/paw; s.c.) produced a dose-related and almost complete inhibition of spermine- induced nociception (1000 nmol/paw, Figure 4A). The calculated mean ID50 value for
this effect (and its respective 95% confidence limit) was 0.24 (0.03-1.76) nmol/paw and the maximal inhibition was 81±10% compared with the control group. As previously described in literature,12,29 the co-administration of capsazepine (1
nmol/paw; s.c.) also largely reduced the capsaicin-induced nociception (1 nmol/paw, Figure 4B), with inhibition of 80±6% compared with the control group. To confirm the hypothesis that the spermine-induced nociception is mediated by TRPV1 receptor, the mice received a co-administration of selective TRPV1 receptor antagonist SB366791 (0.3-10 nmol/paw) with spermine. SB366791 also reduced spermine- induced nociception (Figure 4C), with inhibition of 68±9% compared with the control group and the ID50 was 0.6 (0.3-1.4) nmol/paw. Similarly, the co-administration of
SB366791 (1 nmol/paw) reduced the capsaicin-nociception (Figure 4D), with an inhibition of 72±8% compared with the control group. These data suggest that the activation of TRPV1 receptor is an important mechanism underlying the nociception induced by peripheral injection of spermine in mice.
Participation of TRPV1 positive fibers in the spermine-induced nociception
To investigate the role of TRPV1 expressing afferent fibers on spermine- induced nociception, animals were submitted to a systemic RTX desensitization protocol. The systemic treatment with RTX significantly reduced the expression of TRPV1 protein in the sciatic nerve 7 days after injection, with an inhibition of 67±11%
compared to the control group, confirming a reduction in TRPV1 positive sensory fibers (Figure 5A). We observed that pre-treatment with RTX (50 μg/Kg, s.c., 7 days before) largely reduced the capsaicin (1 nmol/paw) or spermine (1000 nmol/paw)- induced nociception (Figure 5B), with inhibitions of 92±4 and 82±9% compared with the control group, respectively.
Effect of spermine in resiniferatoxin binding and nociception
To clearly verify the interaction between polyamines with TRPV1 receptor, we performed a specific binding assay of RTX in presence or absence of spermine. It was possible to observe that spermine increases the [3H]-RTX specific binding in the
TRPV1 containing membranes, with an IC50 value of 54.0 (43.1 - 67.7) µM and the
maximal effect of 76±5% of specific binding enhancement when compared to the control group (Figure 6A). Again, suggesting that polyamine-induced nociception is mediated by an interaction with TRPV1 receptor, we verified that the co- administration of spermine with RTX, in doses where they alone produced no nociceptive effect, resulted in a significant nociception (Figure 6B). These results strongly reinforce the idea that polyamines induce nociception by facilitating the activation of TRPV1 receptor.
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Natural polyamines (putrescine, spermidine and spermine) are aliphatic amines containing two (putrescine), three (spermidine) or four (spermine) amine groups.14 It is well known that they have an important function in the control of the
neuronal excitability through direct interaction with different ion channels, including glutamate receptors NMDA and AMPA/kainate, ASIC and TRPV1.2,3,35 All these ionic
channels are important to the development and processing of nociception. Since the levels of polyamines are increased in inflammatory processes36 and some drugs
acting on polyamines are in clinical trials to treat hyperproliferative disorders,14,27 the
present study could indicate new targets to treat painful conditions. In our study, we have shown that polyamines induced nociception when injected into the mouse paw by stimulation of the TRPV1 receptor present in small diameter afferent fibers. However, the stimulation of NMDA, AMPA/kainite or ASIC receptors seems to be not important to spermine-induced spontaneous nociception.
We have found that exogenous spermine and spermidine, which are the most charged polyamines, were more potent than putrescine in producing nociception. Our results are in accordance with some data demonstrating that some actions of polyamines in ion channels are charge-dependent.2,35 The pro-nociceptive action of
polyamines found here are also in line with previous findings that show that the feeding of rats with a polyamine-deficient diet may produce analgesic action.11,17,27
Thus, both the restriction of polyamine ingestion and the inhibition of polyamine synthesis could be interesting targets to control painful processes.
It is well known the modulation of ion channels, including ionotropic glutamate receptors, by polyamines.35 In fact, spermine acts at extracellular sites and at pore in
neurons to potentiate the activity of NMDA, AMPA and kainate receptors, respectively. It has been hypothesized that these effects of spermine involve at least
two discrete polyamine-binding sites on NMDA receptors.35 In accordance with these
data, it has been demonstrated that intrathecal (spinal cord) administration of spermine causes hyperalgesia in rats and pain-related behaviors in mice by modulation of the NMDA receptor.19,32 These results indicate that polyamines in
spinal cord can induce nociception through NMDA receptor-stimulation. Furthermore, peripheral administration of NMDA, AMPA/kainite results in painful behavior in rats.37
In contrast with these data, our results demonstrated that the peripheral injection of NMDA receptor antagonist MK 801, in a dose where it is capable of reducing glutamate-induced nociception, failed in altering intraplantar spermine-induced nociception. Thus, we suggest that the peripheral mechanisms involved in spermine- induced nociception are different from the spinal mechanisms. However, the reduction of polyamine-induced nociception by the NMDA antagonists in spinal cord could be indirect since the activation of TRPV1 in spinal cord may release of glutamate in vitro33 and produce an NMDA-mediated nociception in vivo.23 However,
further studies must be carried out to elucidate the role of spinal TRPV1 in the nociceptive action of intrathecally-injected polyamines.
Another ion channel that has been implicated in some polyamine action is the acid sensitive ion channel ASIC-1.3 We found that the non-selective ASIC receptor
blocker amiloride was not able to reduce the spermine-produced nociception in mice in a dose where it reduced acid-induced nociception.20 This result could be explain
because the central nervous system seems to be the major place to the role of ASIC- 1 on nociception.20 Moreover, an in vitro study has showed that spermine shift the
steady-state inactivation curve of ASIC-1 channel.3 Thus, the action of polyamines on
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result indicates that the peripheral ASIC-1 receptor seems not to be a target related to polyamine-induced nociception.
Besides ionotropic glutamate and ASIC receptors, polyamines are also capable of directly stimulating TRPV1, producing ionic selectively changes in this receptor and resulting in Ca2+ influx in sensory neurons.2,9 Furthermore, extracellular
spermine, spermidine and putrescine directly activate and sensitize TRPV1 in a charge-dependent manner, both in heterologous expression systems and in sensory neurons.2 Here, we have shown that both capsazepine and SB366791 largely
reduced spermine-induced nociception, demonstrating a critical role of TRPV1 in the